3.

034 HW#5 Due November 22, 2010

1) An incomplete diagram of genetic material is below.

i) Label the 3' and 5' ends of both strands
ii) Is the double stranded molecule DNA or RNA? How do you know?
iii) Draw in the complementary base pairs
iv) Write out the nucleotide sequence. Be sure to specify which end is 5' and which is 3'
v) Show the mechanism of RNase A. Why or why would it not work on this molecule?
vi) You need to design three different short DNA sequences, 10 base pairs long per strand, that
have three different melting temperatures. Using only single letter designations (i.e., ATGC for each
strand) write out three different sequences that would give three different melting temperatures. Label
the sequences as lowest, mid, and highest melting temperatures. Also, show the strand direction or
polarity for each pair of sequences you write.
2) You have decided to help the Department of Homeland Security design a biological decontamination
surface that has an enzyme strongly linked to a surface that will cleave a particular protein on the
surface of a virus and make the virus inactive for infection.

● The N-terminus of this enzyme has a few lysines followed by a string of glycine.
● The active site is located very close to the C-terminus of the enzyme.
● The protein has one cysteine located near the active site of the enzyme.

It is your job to think about how to attach the enzyme to this surface such that you can wash and reuse
it, and so that the enzyme has good efficiency.

You are provided with aspartic acid terminated gold covered glass, lysine terminated gold covered
glass, and cysteine terminated gold covered glass:

The following molecules are available for coupling, and you can use any other coupling agent if you
wish.

i) Show how you would couple the enzyme to the three surfaces above
ii) Is there one approach (surface and coupling agent) that is preferable to the others? Explain.

3) Gold nanoparticles interact with light, and laser light is a source of energy that can be transmitted to
the location of gold nanoparticles to heat them and cause hyperthermia. This technique has been under
investigation to destroy solid tumors.

Nanoparticles made of semi-conductors can be used for imaging. According to a recent paper, “to take
full advantage of the unique optical properties of quantum dots (Qds) and expedite future near-infrared
fluorescence (NIRF) imaging applications, QDs need to be effectively, specifically and reliably
directed to a specific organ or disease site after systemic adminstration.”

You would like to join three nanoparticles by complementary pairing of DNA.

● The three nanoparticles are made of two different materials

○ Nanoparticles 1 & 2 are made of gold (for thermal ablation)
○ Nanoparticle 3 is made of a semiconducting material (for imaging)
● One of the gold nanoparticles is conjugated to an antibody directed to cancer cells.
i) Provide the sequence of the single-stranded DNA attached to each nanoparticle
ii) Provide the sequence of additional strands that you would need to form a complex of the three
nanoparticles and DNA
iii) Explain why you expect the DNA and nanoparticles to form a complex
iv) Would the color of the gold nanoparticles change upon forming a complex? Explain this effect and
whether there would be an absorption shift towards red or blue.
v) You would like to test quickly whether the particles could be used for thermal ablation. Design an
experiment to determine whether your particles are heating their surroundings.

4) You are going to build a Protease Detector to measure the possible presence of three different
proteases (protease A, B, and C) that are released from a Homeland Security virus decontamination
device.

You have a fully equipped lab and you can order or make any peptide sequence, DNA sequence or
antibody. You have any size gold nanoparticles, gold substrate, glass substrates, carbon nanotubes,
quantum dots, fluorescent dyes or any substrate you want.

Your company wants to use the following platform for detection, and has asked you work out the
details and do the initial testing.

The gold particles may be the same or different sizes. You may add any other molecules to the
particles as needed (but show or explain the attachment chemistry). X is the end of the protein.

i) Design the protein sequences that would be between each gold nanoparticle. List what the
important sequences would be in each region. Explain how you would attach the proteins or
peptides at each attachment point. Assume you can attach a single protein molecule to a gold
nanoparticle. How do you know they are attached?
ii) Attach these nanoparticle protein complexes to a surface. This will be your detection platform
iii) Show (sketch) the nanoparticle pattern for all possible combinations of cleavage, i.e. Only
protease A, only protease B, only protease C, and other combinations.
iv) Design a read out pattern our a way to distinguish between the combinations of proteases, i.e.
To tell what proteases are present.
v) Design a more “traditional” assay to check the validity of your results
vi) Based on your protein design in part a), assign names to proteases A, B, and C